Evaluation dopamine receptors in Parkinson's

描述（申请人提供）：帕金森病（PD）是由黑质纹状体多巴胺能神经元变性引起的。这一过程最终导致多巴胺介导的纹状体信号逐渐减少，表现为一种以运动迟缓、僵硬、震颤和步态异常为特征的临床综合征。PD的传统治疗旨在通过给予多巴胺前体左旋多巴来恢复纹状体中的多巴胺水平。随着疾病的发展，左旋多巴会导致运动障碍和运动活动的显著波动期（“开关效应”）。减轻这些副作用一直是一项重大挑战，并促使人们寻找能够提供更稳定水平的多巴胺能信号传导的替代策略。在恢复纹状体多巴胺能活性的同时，避免长期左旋多巴的潜在后果，一个以前未被探索的选择是通过引入构成活性的多巴胺受体。PI实验室在通过引入激活点突变产生具有配体独立（或组成）活性的受体方面拥有丰富的经验。即使在缺乏多巴胺和/或多巴胺能激动剂的情况下，这些受体也有维持多巴胺能信号传导的潜力。本应用的前提是，组成活性多巴胺受体可以通过体外检测识别，并在大鼠纹状体中表达，以在较长时间间隔内增强多巴胺能信号。Specific Aim 1的目的是在体外产生一系列组成型活性多巴胺1和多巴胺2受体并对其进行药理学表征。利用重组腺相关病毒，我们将在大鼠中探索纹状体过表达构成性活性多巴胺受体的功能后果（Specific Aim 2）。单侧给药后的循环行为将作为构建活性的指标。所使用的方法将包括分子（产生构成活性突变受体，表达重组蛋白），药理学（放射寡配结合，第二信使信号分析）和行为方法（评估循环行为）。这些实验将为纹状体中多巴胺能受体的作用提供更多的见解，并有可能为帕金森病的新治疗选择的发展迈出第一步。

DESCRIPTION (provided by applicant): Parkinson's disease (PD) results from the degeneration of nigrostriatal dopaminergic neurons. This process ultimately leads to a progressive decrease in dopamine mediated striatal signaling which manifests as a clinical syndrome characterized by bradykinesia, rigidity, tremor, and gait abnormalities. Traditional therapy for PD has aimed at restoring dopamine levels in the striatum through administration of the dopamine precursor, L-dopa. With advanced disease, L-dopa leads to dyskinesias and periods of marked fluctuation in motor activity ('on-off effect'). Alleviation of these side effects has been a major challenge and has prompted a search for alternative strategies which can provide a more stable level of dopaminergic signaling. A previously unexplored option to restore striatal dopaminergic activity and at the same time to potentially avoid the consequences of long term L-dopa administration, is through the introduction of constitutively active dopamine receptors. The laboratory of the PI has extensive experience in generating receptors with ligand independent (or constitutive) activity through the introduction of activating point mutations. These receptors have the potential to maintain dopaminergic signaling even in the absence of dopamine and/or dopaminergic agonist drugs. The premise of this application is that constitutively active dopamine receptors can be identified using in vitro assays and expressed in the striatum of rats to enhance dopaminergic signaling over an extended time interval. The objective of Specific Aim 1 is to generate and pharmacologically characterize in vitro a series of constitutively active dopamine 1 and dopamine 2 receptors. Using recombinant adeno-associated virus, the functional consequences of striatal overexpression of constitutively active dopamine receptors will be explored in rats (Specific Aim 2). Circling behavior after unilateral viral administration will be used as an index of construct activity. The methodologies utilized will include molecular (generation of constitutively active mutant receptors, expression of recombinant proteins), pharmacologic (radioligand binding, second messenger signaling assays), and behavioral approaches (assessment of circling behavior). These experiments will provide additional insight into the role of dopaminergic receptors in the striatum as well as potentially take the first steps toward the development of a new therapeutic option for Parkinson's disease.